Adjuvant compositions and methods of use

ABSTRACT

This disclosure provides adjuvant compositions that are capable of modulating the immune response in a subject. These adjuvant compositions may also be used enhance the immunogenicity of antigens. Also provided are methods of making the adjuvant compositions as well as methods of using the adjuvant compositions.

CROSS REFERENCED TO RELATED APPLICATION

This application is a continuing application of U.S. patent applicationSer. No. 12/651,975, filed Jan. 4, 2010, which claims priority to U.S.Provisional Application No. 61/204,316 filed Jan. 5, 2009.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.1R43AI084690-01 awarded by the National Institute of Health. Thegovernment has certain rights in this invention.

TECHNICAL FIELD

This present disclosure relates to adjuvant compositions and methods forusing the same.

BACKGROUND

Vaccines are very cost effective medical interventions. However,although many diseases have been prevented through vaccinations, manyothers remain to be prevented. Moreover, improved vaccines are neededfor a number of diseases for which vaccines already exist. A majorhurdle in producing vaccines is the lack of or low immunogenicity of thevaccine. The effectiveness of a vaccine can be enhanced by usingadjuvants and delivery systems. Of particular interest are adjuvantsthat increase the immunogenicity of a vaccine administered by aneedle-free method via a mucosal route.

SUMMARY

This disclosure provides adjuvant compositions that are capable ofmodulating the immune response in a subject. These adjuvant compositionsmay also be used enhance the immunogenicity of antigens by enhancingantigen-presentation, enhancing innate immune responses throughactivation of, e.g., natural killer cells, and/or direct B cellactivation. Also provided are methods of making the adjuvantcompositions as well as methods of using the adjuvant compositions.

In certain embodiments, the compositions include mustard oil; and atleast one of: (a) a flavonoid, or a flavonoid derivative, or a flavonoidderivative salt; and (b) a vitamin, or a derivative or salt thereof,where the vitamin is selected from the group consisting of vitamin A,vitamin E, vitamin D, vitamin C. In certain cases, the compositionsinclude the mustard oil and the flavonoid, such as, a catechin, or theflavonoid derivative, or the flavonoid derivative salt. In certaincases, the flavonoid is a catechin, or a derivative or a salt thereof.In certain cases, the composition comprises the mustard oil and thevitamin, or a derivative or salt thereof. In certain embodiments, thecomposition includes mustard oil, the flavonoid, or the flavonoidderivative, or the flavonoid derivative salt and the vitamin, or aderivative or salt thereof. In certain embodiments, the compositionincludes the mustard oil and the vitamin may be vitamin E, or aderivative or salt thereof. In certain embodiments, the compositionincludes the mustard oil and vitamin A, or a derivative or salt thereof.In certain embodiments, the composition includes the mustard oil andvitamin C, or a derivative or salt thereof. In certain embodiments, thecomposition includes the mustard oil, a catechin, or a derivative orsalt thereof, and vitamin A, or a derivative or a salt thereof. Incertain embodiments, the composition includes the mustard oil, acatechin, or a derivative or salt thereof, and vitamin E, or aderivative or a salt thereof. In certain embodiments, the compositionincludes the mustard oil, the flavonoid, such as a catechin, or theflavonoid derivative, or the flavonoid derivative salt, and allylisothiocyanate. In certain embodiments, the composition includes themustard oil, the vitamin, such as vitamin A, or a derivative or saltthereof, and allyl isothiocyanate. In certain embodiments, thecomposition includes the mustard oil, the flavonoid, such as catechin,or the flavonoid derivative, or the flavonoid derivative salt, thevitamin, such as vitamin A, or a derivative or salt thereof, and allylisothiocyanate. In certain aspects, the composition further includes anantigen. In certain embodiments, the adjuvant compositions include allylisothiocyanate and at least one of: a flavonoid and a vitamin.

The compositions may be administered to a subject, such as a mammal, bya number of routes, such as, intranasal, pulmonary, sublingual, oral,buccal, intra-vaginal, intra-rectal, ocular, intradermal, transdermal,transcuataneous, subcutaneous, intra-venous and intramuscular.

Also provided are methods for making the compositions, the methodincludes admixing of the mustard oil and at least one of the flavonoid,or the flavonoid derivative, or the flavonoid derivative salt; and thevitamin, or derivative or salt thereof.

Also provided herein are compositions that include a pharmaceuticallyacceptable vegetable oil; a flavonoid, or a flavonoid derivative, or aflavonoid derivative salt; and a vitamin, or a vitamin derivative, or avitamin derivative salt, where the vitamin is selected from the groupconsisting of vitamin A, vitamin E, vitamin D, vitamin C. In certaincases, the compositions include the vegetable oil and the flavonoid,such as, a catechin, or the flavonoid derivative, or the flavonoidderivative salt. In certain cases, the flavonoid is a catechin, or aderivative or a salt thereof. In certain cases, the compositioncomprises the vegetable oil and the vitamin, or a derivative or saltthereof. In certain embodiments, the composition includes vegetable oil,the flavonoid, or the flavonoid derivative, or the flavonoid derivativesalt and the vitamin, or a derivative or salt thereof. In certainembodiments, the composition includes the vegetable oil and the vitaminmay be vitamin E, or a derivative or salt thereof. In certainembodiments, the composition includes the vegetable oil and vitamin A,or a derivative or salt thereof. In certain embodiments, the compositionincludes the vegetable oil and vitamin C, or a derivative or saltthereof. In certain embodiments, the composition includes the vegetableoil, a catechin, or a derivative or salt thereof, and vitamin A, or aderivative or a salt thereof. In certain embodiments, the compositionincludes the vegetable oil, a catechin, or a derivative or salt thereof,and vitamin E, or a derivative or a salt thereof. In certainembodiments, the composition includes the vegetable oil, the flavonoid,such as a catechin, or the flavonoid derivative, or the flavonoidderivative salt, and allyl isothiocyanate. In certain embodiments, thecomposition includes the vegetable oil, the vitamin, such as vitamin A,or a derivative or salt thereof, and allyl isothiocyanate. In certainembodiments, the composition includes the vegetable oil, the flavonoid,such as catechin, or the flavonoid derivative, or the flavonoidderivative salt, the vitamin, such as vitamin A, or a derivative or saltthereof, and allyl isothiocyanate. In certain aspects, the compositionfurther includes an antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows serum titers of anti-HA IgG1 antibody and anti-influenza-HAIgG2a antibody at three-weeks after single intramuscular (IM)vaccination.

FIG. 2 shows serum titers of anti-influenza-HA IgG1 antibody and anti-HAIgG2a antibody at one-week after the second intramuscular (IM)vaccination.

FIG. 3 shows serum titers of anti-influenza-HA IgA antibody at one-weekafter the second intramuscular (IM) vaccination.

FIG. 4 shows titers of anti-influenza-HA IgG1 antibody in vaginal lavageat one-week after the second intramuscular (IM) vaccination.

FIG. 5 shows titers of anti-influenza-HA IgA antibody in vaginal lavageat one-week after the second intramuscular (IM) vaccination.

FIG. 6 shows TH1, TH2 and Treg responses following ex vivo activationwith influenza-HA at one-week after the second intramuscular (IM)vaccination.

FIG. 7 shows serum titers of anti-HIVgp120 IgG1 antibody andanti-HIVgp-120 IgG2a antibody at two weeks after the second intranasal(IN) vaccination.

FIG. 8 shows titers of anti-HIVgp120 IgG1 antibody and anti-HIVgp120 IgAantibody in vaginal lavage at two weeks after the second IN vaccination.

FIG. 9 shows serum titers of anti-HIVgp120 IgG1, IgG2a, and IgAantibodies at one week after two IN/SL (Intranasal/sublingual) and twoIM vaccinations.

FIG. 10 shows titers of anti-HIVgp120 IgG1 and IgA antibodies in vaginallavage at one week after two IN/SL (Intranasal/sublingual) and two IMvaccinations.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. To the extent such publications may set out definitions of a termthat conflicts with the explicit or implicit definition of the presentdisclosure, the definition of the present disclosure controls.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anadjuvant composition” includes a plurality of adjuvant composition,reference to “a vitamin” includes one, two, or more vitamins, andreference to “a flavonoid” includes one, two, or more flavonoids, and soforth. The terms adjuvant and delivery system may be usedinterchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present disclosure provides adjuvant compositions that are capableof modulating the immune response in a subject. These adjuvantcompositions may also be used enhance the immunogenicity of antigens byenhancing antigen-presentation, enhancing innate immune responsesthrough activation of, e.g., natural killer cells, and/or direct B cellactivation. Also provided are methods of making the adjuvantcompositions as well as methods of using the adjuvant compositions.

DEFINITIONS

The phrases “adjuvant composition(s)” refer to a composition that whenadministered to a subject is capable of inducing an immune response inthe subject. When administered in combination with an antigen, the“adjuvant compositions” are capable of eliciting an antigen-specificimmune response.

An “immune response” to an antigen or composition is the development ina subject of a humoral and/or a cellular immune response to moleculespresent in the antigen or composition of interest. A “humoral immuneresponse” refers to an immune response mediated primarily by antibodymolecules, while a “cellular immune response” is one mediated primarilyby T-lymphocytes and/or other white blood cells.

The phrase “pharmaceutically acceptable” refers to substance that isgenerally safe and is acceptable for veterinary use when the subject isa non-human. “Pharmaceutically acceptable” for humans refers tosubstance that is generally safe and is acceptable for humanpharmaceutical use.

The term “antigen” refers to any substance that can be recognized by theimmune system (e.g., bound by an antibody or processed so as to elicit acellular immune response by, e.g. T cells) under appropriate conditions.An antigen contains one or more epitopes. A B-cell epitope includes atleast about 3-5 amino acids, for example, 4 or more amino acids. Ahapten or a polysaccharide may also serve as a B cell epitope. A T-cellepitope, such as a cytotoxic T-cell (CTL) epitope, may include at leastabout 7-9 amino acids, for example, 8 or more amino acids. A helperT-cell epitope may include at least about 12-20 amino acids. The term“antigen” denotes both subunit antigens (i.e., antigens which areseparate from the whole organism with which the antigen is associated innature), as well as, killed, attenuated or inactivated bacteria,viruses, fungi, parasites or other microbes, prions, allergens or anyother disease causing agents. An antigen may be a modified protein thatincludes modifications, such as deletions, additions and substitutions(generally conservative in nature) to the native protein sequence. Theterm antigen also denotes nucleic acids (DNA or RNA) encoding a proteinor peptide antigen.

“Penetration enhancement” or “permeation enhancement” as used hereinrefers to increasing the permeability of skin or mucosa to an antigen soas to increase the rate at which the antigen passes through the skin ormucosa and enters the lymph node or the blood stream.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for preventing or treating a disease, is sufficient to affectsuch prevention or treatment for the disease. The “therapeuticallyeffective amount” will vary depending on the compound, the disease andits severity and the age, weight, etc., of the subject to be treated.

Overview

This disclosure provides adjuvant compositions that are capable ofinducing an immune response. These adjuvant compositions may also beused enhance the immunogenicity of antigens by enhancingantigen-presentation, enhancing innate immune responses throughactivation of, e.g. natural killer cells, and/or by direct B cellactivation. Also provided are methods of making the adjuvantcompositions as well as methods of using the adjuvant compositions.

Adjuvant Compositions

Adjuvant compositions provided herein include: a pharmaceuticallyacceptable vegetable oil; a flavonoid, or a flavonoid derivative, or aflavonoid derivative salt; and a vitamin, or a derivative, or a saltthereof. The vitamin may be vitamin A, vitamin E, vitamin D, vitamin C.

In another embodiment, the compositions provided herein include: mustardoil with or without allyl isothiocyanate (essential oil of mustard); andat least one of: (a) a flavonoid, or a flavonoid derivative, or aflavonoid derivative salt; and (b) a vitamin, or a derivative, or a saltthereof. The vitamin may be vitamin A, vitamin E, vitamin D, vitamin C,or a derivative or a salt thereof.

In certain embodiments, the flavonoid is a flavone, a flavonol, aflavonone, a catechin, anthocyanid, or isoflavone, or derivativesthereof, or salts of the derivatives. In certain embodiments, theadjuvant composition includes mustard oil and a catechin, such as,catechin hydrate.

In certain embodiments, the adjuvant compositions include one or morevitamins, such as, Vitamin A, Vitamin E, Vitamin D, Vitamin C,derivatives, and salts thereof and a vegetable oil carrier, such asmustard oil. The adjuvant compositions may optionally include aflavonoid or a flavonoid derivative or salt of the flavonoid derivative.In certain embodiments, the adjuvant compositions include Vitamin A andan oil carrier, such as, mustard oil. In certain embodiments, theadjuvant compositions include Vitamin E and an oil carrier, such as,mustard oil.

The adjuvant compositions may additionally include other additives, suchas preservatives, colorants, flavorants, etc. The adjuvant compositionsmay additionally include an antigen.

Pharmaceutically Acceptable Vegetable Oil Carriers

A “pharmaceutically acceptable vegetable oil carrier” as used hereinrefers to a vegetable oil that is suitable for administration to a humanor non-human animal by a desirable route, e.g., systemic or mucosalroute, including oral and topical routes of delivery. Edible adjuvantcompositions are contemplated by the present disclosure.

“Vegetable oil” refers to oil obtainable from a plant or a plantproduct, and encompasses oil obtainable from seeds (including nuts,grains), fruits, roots, flowers, stems, etc. Examples include corn oil,mustard oil, olive oil, coconut oil, safflower oil, soybean oil, and thelike. Vegetable oils of the present disclosure encompass oils obtainablefrom non-genetically modified and from genetically modified plants.Vegetable oils encompass vegetable oils having properties of arubefacient, i.e., oils that promotes dilation of capillaries and anincrease in blood circulation, e.g., when applied topically to skin.Vegetable oil may be derived from a plant or plant product (e.g., anon-genetically modified or genetically modified plant or plantproduct), or may be produced synthetically, e.g., by mixing theindividual components found in vegetable oils, where the individualcomponents may be derived from plants or plant products, or producedsynthetically. The plants which provide the source for the vegetable oilor the individual fatty acids may be genetically modified.

In certain embodiments, the vegetable oil is a mustard oil. “Mustardoil” as used herein refers to oil that is obtainable from seeds of amustard plant of Brassicacae, where the oil is obtainable from themustard plant without application of heat during extraction (e.g.,obtainable by a cold-press extraction method). Mustard oil obtainablefrom seeds of a mustard plant without application of heat have a loweramounts of (e.g., no significant or detectable) allyl isothiocyanatethan oil that may be obtainable from the same seeds using a heat-basedextraction method (e.g., by application of steam). Mustard plants ofBrassicacae from which mustard oils useful as carriers in thecompositions of the present disclosure may be obtainable include, butare not necessarily limited to, Brassica rapa (edible greens), Brassicanigra (black mustard), Brassica juncea (brown mustard), Brassica hirta(white or yellow mustard), Brassica carinata (Ethiopian mustard),Brassica oleracea (wild mustard), Brassica campestris (includingBrassica napus L. and B. campestris L.), and Brassica napus. Oilscontemplated by “mustard oil” can include oil obtainable from rapeseed.

In certain embodiments, the vegetable oil is canola oil. Such canola oilmay have the following composition: 6-8% Saturated Fatty Acids (with 3.5Palmitic Acid); 14.4% Monounsaturated Fatty Acids (with 60% Oleic Acid);and 69.3% Polyunsaturated Fatty Acids (with 20% Linoleic Acid, 10% AlphaLinolenic Acid).

In certain embodiments, the vegetable oil used in the compositionsdescribed herein may be composed of about 14%-70% monounsaturated fattyacids, about 18%-22% polyunsaturated fatty acids and about 5%-12%saturated fatty acids. The monounsaturated fatty acids may have about18%-51% erucic acid and about 7%-22% oleic acid, the polyunsaturatedfatty acids may have about 9-15% linolenic acid and about 6-24% linoleicacid, and the saturated fatty acids may have about 3-4% palmitic acid.

In certain embodiments, the vegetable oil used in the compositionsdescribed herein may be composed of 14%-70% monounsaturated fatty acids,18%-22% polyunsaturated fatty acids and 5%-12% saturated fatty acids.

In certain embodiments, the vegetable oil used in the compositionsdescribed herein may be composed of 14%-20% monounsaturated fatty acids,18%-20% polyunsaturated fatty acids and 5%-6% saturated fatty acids.

In certain embodiments, the vegetable oil used in the compositionsdescribed herein may be composed of about 60%-70% monounsaturated fattyacids, about 18%-22% polyunsaturated fatty acids and about 5%-6%saturated fatty acids.

Where the vegetable oil is a mustard oil, in certain embodiments, themustard oil may have the following composition: monounsaturated fattyacids (erucic acid (18-51%), oleic acid (7-22%)), polyunsaturated fattyacids (linolenic (9-15%) and linoleic (6-24%)), and 5% saturated fattyacids. The mustard oil may additionally also include other components,such as, proteins (30%), phenolics, phytin and dithiol thiones. Mustardoil may also contain 490 mg/100 gm of calcium. Mustard oil may alsocontain 9-15% omega 3 fatty acids.

In some embodiments, the mustard oil is one obtainable from Brassicarapa. Mustard oil obtainable from Brassica rapa includes an oil havingthe following composition: 5.4% Saturated Fatty Acids (with 2.7%Palmitic Acid, 1.0% Stearic Acid, 0.6% Behenic, 1.1% Other); 67.3%Monounsaturated Fatty Acids (with 23.3% Oleic, 10.0% Gadoleic, 33.8%Erucic); and 20.6% Polyunsaturated Fatty Acids (with 9.4% Linoleic Acid,9.9% Alpha Linolenic Acid).

In certain embodiments, the vegetable oil carrier (e.g., mustard oil)may be a mixture of one or more vegetable oils, for example, mustard oil(with or without added AIT) and corn oil; mustard oil (with or withoutadded AIT) and soy bean oil; mustard oil (with or without AIT) andcoconut oil. The present disclosure also contemplates compositionshaving a vegetable oil carrier that itself is a rubifacient and/orcombined with a rubefacient oil. Examples of rubefacient oils includeOil of Wintergreen (Methyl Salicylate), mustard oil, and Rosemary oil(Rosmarinus officinalis).

Flavonoids

The adjuvant compositions may include one or more flavonoids orderivates or salts thereof. Flavonoids (also known as bioflavonoids) arephytochemicals found in fruits and vegetables. Flavonoids are of thefollowing types: Flavones (e.g., apigenin, luteolin), Flavonols (e.g.,quercetin, myricetin), Flavanones (e.g., naringenin, hesperidin),Catechins (e.g., epicatechin, catechin, epigallocatechin, gallocatechin,epicatechin gallate and epigallocatechin gallate),Anthocyanidins/anthocyanins (e.g., cyanidin, pelargonidin), andIsoflavones (e.g., genistein, daidzein).

In certain embodiments, the adjuvant compositions may includeepigallocatechin gallate (EGCG), a form of catechin (polyphenols). Insome embodiments, the adjuvant compositions may include a catechin, suchas, catechin hydrate. In some embodiments, the catechin is not amultimeric form of catechin.

Vitamins

The adjuvant compositions may optionally include one or more vitamins,or derivatives or salts thereof. The one or more vitamins may be one ormore of vitamin A, vitamin E, vitamin D, vitamin C, and derivatives andsalts thereof.

Vitamin A.

Vitamin A is a fat-soluble vitamin that is derived from two sources:preformed retinoids and provitamin carotenoids. Retinoids, such asretinal and retinoic acid, are found in animal sources like liver,kidney, eggs, and dairy produce. Carotenoids like beta-carotene (whichhas the highest vitamin A activity) are found in plants such as dark oryellow vegetables and carrots. Vitamin A is also known as retinol,retinoic acid, Axerophthol, Vitamin A alcohol, Vitamin A1,all-trans-3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraen-1-ol.In certain cases, an acid derivation of Vitamin A, all-trans retinoicacid (ATRA), may be included in the adjuvant compositions. In certaincases, the adjuvant compositions may include retinoids, for example,isotretinoin (Ro 4-3780), etretinate (RO 10-9359; a synthetic retinoid),or motretinide (Ro 11-1430). In certain cases, vitamin A palmitate (VA)may be included in the adjuvant compositions.

Vitamin E.

Of the eight natural substances exerting vitamin E activity (α-, β-, δ-,and γ-tocopherols and α-, β-, δ-, and γ-tocotrienols), α-tocopherol(α-T) has traditionally been regarded as the most important vitaminbecause it exerts the highest biological activity of all vitamins whenassessed in animal model systems. Vitamin E is also synonymous with(±)-α-Tocopherol and DL-all-rac-α-Tocopherol, 5,7,8-Trimethyltocol,D-α-Tocopherol,2,5,7,8-Tetramethyl-2-(4′,8′,12′-trimethyltridecyl)-6-chromanol, and anon-oxidizing version (±)-α-Tocopherol acetate and the relatedall-rac-α-Tocopheryl acetate. A related molecule is D-α-Tocopherolsuccinate, Vitamin E succinate.

In certain embodiments, the vitamin E included in the adjuvantcompositions may be α-tocopherol (α-T). In other cases, a vitamin Eanalog, such as Alpha tocopheryl succinate (alpha-TOS) may be includedin the adjuvant compositions.

Vitamin D.

Vitamin D is a group of fat-soluble prohormones, the two major forms ofwhich are vitamin D₂ (or ergocalciferol) and vitamin D₃ (orcholecalciferol). Vitamin D obtained from sun exposure, food, andsupplements, is biologically inert and must undergo two hydroxylationreactions to be activated in the body. Calcitriol(1,25-Dihydroxycholecalciferol) is the active form of vitamin D found inthe body. The term vitamin D refers to these metabolites and otheranalogues of these substances. In certain embodiments, the adjuvantcompositions may include 1,25-Dihydroxyvitamin D₃ (DHVD₃).

Vitamin C.

Vitamin C or L-ascorbic acid is an essential nutrient for humans.Ascorbate (an ion of ascorbic acid) is required for a range of essentialmetabolic reactions in all animals and plants. The pharmacophore ofvitamin C is the ascorbate ion. In living organisms, ascorbate is ananti-oxidant, since it protects the body against oxidative stress, andis a cofactor in several vital enzymatic reactions. Vitamin C is purelythe L-enantiomer of ascorbate; the opposite D-enantiomer has nophysiological significance.

Additives

In certain embodiments, the vegetable oil carrier of the adjuvantcomposition may include allyl isothiocyanate (AIT). Allyl isothiocyanate(AIT) is also referred to as volatile oil of mustard or essential oil ofmustard or oil of mustard. AIT is an organosulfur compound of theformula CH₂CHCH₂NCS. AIT is responsible for the pungent taste ofmustard, horseradish, and wasabi. It is slightly soluble in water, butwell soluble in most organic solvents. Allyl isothiocyanate comes fromthe seeds of black or brown Indian mustard. When these mustard seeds arebroken, the enzyme myrosinase is released and acts on a glucosinolateknown as sinigrin to give allyl isothiocyanate. Allyl isothiocyanateserves the plant as a defense against herbivores; since it is harmful tothe plant itself, it is stored in the harmless form of theglucosinolate, separate from the myrosinase enzyme. When an animal chewsthe plant, the allyl isothiocyanate is released, repelling the animal.Allyl isothiocyanate is produced commercially by the reaction of allylchloride and potassium thiocyanate: CH₂═CHCH₂Cl+KSCN→CH₂═CHCH₂NCS+KCl.The product obtained in this fashion is sometimes known as syntheticmustard oil. Allyl isothiocyanate can also be liberated by drydistillation of the seeds. The product obtained in this fashion is knownas volatile oil of mustard and is usually around 92% pure. It is usedprincipally as a flavoring agent in foods. Synthetic allylisothiocyanate is used as an insecticide, bacterialcide, and nematocide,and is used in certain cases for crop protection.

In certain cases, the adjuvant composition may be composed of: AIT and avitamin (such as, one ore more of the vitamins A, C, D, E, or salts orderivatives thereof). In certain cases, the adjuvant composition may becomposed of: AIT and a flavonoid (such as a catechin, for example,catechin hydrate). In certain cases, the adjuvant composition may becomposed of: AIT, a vitamin (such as, one ore more of the vitamins A, C,D, E, or salts or derivatives thereof), and a flavonoid (such as acatechin, for example, catechin hydrate).

The adjuvant compositions may include MF59 that contains squalene, aterpenoid plant derivative which has been shown adjuvant properties inanimal and human studies. However, in certain embodiments MF59 orsqualene may not be included in the adjuvant compositions. Thus, in someembodiments, the adjuvant compositions provided herein include aflavonoid; a pharmaceutically acceptable oil carrier; and optionally oneor more vitamins selected from the group consisting of Vitamin A,Vitamin E and Vitamin C but does not include MF59 or squalene.

The adjuvant compositions may include saponin and its derivative QS-21.However, in certain embodiments saponin may not be included in theadjuvant compositions. Thus, in some embodiments, the adjuvantcompositions provided herein include a flavonoid; a pharmaceuticallyacceptable oil carrier; and optionally one or more vitamins selectedfrom the group consisting of Vitamin A, Vitamin E and Vitamin C but doesnot include saponin or its derivatives.

Derivatives of phytol, a dietary diterpene alcohol, similar in structureto naturally occurring isoprenoid adjuvants, elicit increased titers ofall major IgG subclasses, especially IgG2a and cytotoxic effector T cellresponses. The adjuvant compositions may include phytol or itsderivates. However, in certain embodiments phytol may not be included inthe adjuvant compositions. Thus, in some embodiments, the adjuvantcompositions provided herein include a flavonoid; an oil carrier; andoptionally one or more vitamins selected from the group consisting ofVitamin A, Vitamin E and Vitamin C but does not include phytol or itsderivatives.

The adjuvant compositions may include other additives, such as, gelatin,antibiotics, sorbitol, sucrose, lactose, other sugars, bioadhesives,mucoadhesives (e.g., hyaluronic acid or a derivative thereof, forexample, HYAFF), hydrophilic polymers and hydrogels, polyethylene oxidehomopolymers, chitosan, Beeswax, and the like.

The adjuvant compositions may include immunogenicity enhancing agents,such as, lipopolysaccharides, enterotoxins such as the heat labile toxinfrom Escherichia coli bacterium, cholera toxin from Vibrio cholerae,toll like receptor agonists (e.g., CpG or CpG oligonucleotides). Theadjuvant compositions may be combined with other vaccine deliverysystems, such as, alum, liposomes, oil-in-water emulsions, for example.

The adjuvant compositions may be formulated with large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Examples of particulate carriers includethose derived from polymethyl methacrylate polymers, as well asmicroparticles derived from poly(lactides) and poly(lactide coglycolides), known as PLG.

The adjuvant compositions may include The mutant forms of a holotoxin,e.g. from E. coli, comprising the mutated A subunit and the B subunit,which may be oligomeric, as in the wild-type holotoxin. The B subunit ispreferably not mutated. However, it is envisaged that a mutated Asubunit may be used in isolation from the B subunit, either in anessentially pure form or complexed with other agents, which may replacethe B subunit and/or its functional contribution. LT mutants for use inthe compositions include mutants with one or more of the followingmutations: a mutation in the A subunit of the serine at position 63, anda mutation in the A subunit of the alanine at position 72, for example,the serine at position 63 is replaced with a lysine and the alanine atposition 72 is replaced with arginine.

The adjuvant compositions may include may include cholera toxin (“CT”)or detoxified mutants thereof and microparticles (i.e., a particle ofabout 100 nm to about 150 μm in diameter, more preferably about 200 nmto about 30 μm in diameter, and still more preferably about 500 nm toabout 10 μm in diameter) formed from materials that are biodegradableand non-toxic (e.g., a poly(.alpha.-hydroxy acid), a polyhydroxybutyricacid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.).

The adjuvant compositions disclosed herein may be formulated asmicroparticle using a poly(α-hydroxy acid), in particular, from apoly(lactide) (“PLA”), a copolymer of D,L-lactide and glycolide orglycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered antigen. If the composition includes an antigen,the antigen may be entrapped within the microparticles, or may beadsorbed onto their surface.

In certain embodiments, the compositions disclosed herein include, animmuno-modulatory factor, for example, a protein that is capable ofmodulating an immune response. Non-limiting examples of immunomodulatoryfactors include lymphokines (also known as cytokines), such as IL-6,TGF-beta, IL-1, IL-2, IL-3, etc.); and chemokines (e.g., secretedproteins such as macrophage inhibiting factor). Certain cytokines, forexample TRANCE, flt-3L, and a secreted form of CD40L are capable ofenhancing the immunostimulatory capacity of APCs. Non-limiting examplesof cytokines which may be used alone or in combination in thecompositions disclosed herein include, interleukin-2 (IL-2), stem cellfactor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12(IL-12), G-CSF, granulocyte macropliage-colony stimulating factor(GM-CSF), interleukin-1 alpha (IL-1.alpha.), interleukin-11 (IL-11),MIP-1.gamma., leukemia inhibitory factor (LIF), c-kit ligand,thrombopoietin (TPO), CD40 ligand (CD40L), tumor necrosis factor-relatedactivation-induced cytokine (TRANCE) and flt3 ligand (flt-3L).

The adjuvant compositions may include emulsifiers, such as, lecithin,for example phospholipids and/or surfactants that are amphiphilic andacceptable for human and/or veterinary use. The surfactants may be ionic(e.g. Tween 80), cationic (e.g. CTAB) or zwitterionic (e.g. CHAPS). Theacceptability of a surfactant for human and/or veterinary use may bedetermined by those of skill in the art. A surfactant is amphiphilic ifa part of the surfactant molecule is hydrophobic and a part ishydrophilic. Examples of surfactants useful in the adjuvant compositionsdisclosed herein include, but are not limited to, a Tween surfactant anda Span surfactant. Tween and Span surfactants include, but are notlimited to, monolaureate (Tween 20, Tween 21, Span 20), monopalmitate(Tween 40, Span 40), monostearate (Tween 60, Tween 61, Span 60),tristearate (Tween 65, Span 65), monooleate (Tween 80, Tween 81, Span80) and trioleate (Tween 85, Span 85).

The adjuvant compositions may include pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, and the like, for example,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate, phosphate buffer saline, and the like.

The adjuvant compositions may include medicinal rubefacients, such as,Capsaicin (derived from Cayenne, Capsicum minimum), Salicylates (such asOil of Wintergreen, Methyl Salicylate), Nicotinate esters, Rubbingalcohol, common herbal rubefacients include: Cloves (Eugeniacaryphyllus), Garlic (Allium sativum), Ginger (Zingiber officinale),Horseradish (Cochlearia armoracia), Mustard (e.g., Brassica alba or B.nigra), Nettle (Urtica dioica), Rosemary Oil (Rosmarinus officinalis),Rue (Ruta graveolens).

Antigens

The adjuvant compositions disclosed herein may be administered incombination with one or more antigens. Any antigen against which it isdesirable to induce an immune response may be used. Such an antigen maybe obtainable from virus, fungi, or bacteria or other human and/oranimal pathogens, or cancer cells. The antigen may be an allergen. Fulllength protein or a fragment thereof as well as modified or unmodifiedprotein may be used as antigen. Antigens also include polysaccharides.In some cases, the antigen may be encoded by a DNA or RNA, e.g., derivedfrom a pathogen or cancer cells.

Cancer Antigens.

Many human cancers express cell surface molecule that are specific tothe cancer cell, i.e., they are not expressed at a detectable level or asignificant level by normal human somatic cells. Examples of suchantigens include but are not limited to the following: variousglycolipids and polysaccharides, Alpha-fetoprotein (AFP) and CancerAntigens CA125, CA15-3, and CA19-9.

AFP:

Elevation of serum AFP to abnormally high values occurs in severalmalignant diseases—including nonseminomatous testicular cancer andprimary hepatocellular carcinoma—and some benign ones, includinghepatitis and cirrhosis.

CA125:

Cancer Antigen 125 (CA125) is a surface antigen associated withepithelial ovarian cancer, and to date CA125 is the most sensitivemarker for residual epithelial ovarian cancer. CA125 may also beelevated in patients with lung, cervical, fallopian tube, and uterinecancer and endometriosis.

CA15-3:

Cancer antigen 15-3 (CA15-3) is useful for monitoring breast cancerpatients post-operatively for recurrence, particularly metastaticdiseases. CA15-3 has been shown to be useful in early detection ofrelapse of ovarian cancer. CA15-3 levels are also increased in colon,lung, and hepatic tumors.

CA19-9:

Serum CA19-9 level is frequently elevated in subjects with certaingastrointestinal malignancies, such as pancreatic, colorectal, gastricand hepatic carcinomas. A persistently rising serum CA 19-9 value may beassociated with progressive malignant disease and poor therapeuticresponse. A declining CA 19-9 value may be indicative of a favorableprognosis and good response to treatment.

Prion Antigens.

Transmissible spongiform encephalopathies (TSEs) are a group ofneurodegenerative diseases characterized by a rapidly progressivedeterioration (in cognitive function and/or coordination) which alwaysleads to death. TSEs occur in humans and in animals. The most likelycause of the TSEs is the prion protein form designated PrPSc, namedafter scrapie, the oldest known form of prion disease, which originatedin sheep and goats. How prions cause brain damage is unclear at present,but all hypotheses suggest that posttranslational modification of thenative prion protein (PrPC) by PrPSc to form amyloid fibrils is acentral event in pathogenesis.

In humans, Creutzfeldt-Jakob disease (CJD) is the most widespread TSE(incidence 1/million/year). Clinically, patients can be diagnosed aspossible or probable CJD patients but neuropathological conformation isnecessary to obtain a definite diagnosis. Neuropathologicalinvestigation is based on a triad of histological lesions: spongiosis,neuron loss, and reactive astrogliosis.

The prion protein (PrP) was initially described as an essentialcomponent of the infectious agents responsible for transmissiblespongiform encephalopathies (TSE). TSE are a group of neurodegenerativedisorders that include Creutzfeldt-Jakob disease and kuru in humans,bovine spongiform encephalopathy, sheep scrapie, and chronic wastingdisease in deer and elk. Although the pathophysiology of TSE remainspoorly understood, an almost invariable feature is the accumulation ofan abnormal isoform of PrP (scrapie PrP, designated PrPSc) in infectedtissues of affected individuals. PrP was found to be encoded by a uniquegene of the host, Prnp (PRNP in humans), the structure of which isremarkably conserved between species. Its physiological product isexpressed as a GPI-anchored membrane protein termed cellular PrP (PrPC),in many tissues at variable levels.

Pathogens.

The antigens for use in combination with the adjuvant compositionsdescribed herein include antigens derived from any pathogens includingviruses, bacteria or fungi, or cancers. Such antigens include, forinstance, the structural as well as nonstructural proteins of apathogen, such as Env, Gag and Pol of HIV or F protein of RSV, or HA ofinfluenza, in their native form or in a form optimized for enhancedimmunogenicity.

Other antigens which may be included in the adjuvant compositions are: Aprotein antigen from N. meningitidis serogroup B, such as those inInternational patent application publications: WO99/24578; WO99/36544;WO99/57280; WO00/22430; and WO96/29412, for example; an outer membranevesicle (OMV) preparation from N. meningitidis serogroup B, such asthose disclosed in. International patent application WO0152885; anoligosaccharide antigen from N. meninigitidis serogroup A, C, W135and/or Y; A saccharide antigen from Streptococcus pneumoniae, an antigenfrom hepatitis A virus, such as inactivated virus, an antigen fromhepatitis B virus, such as the surface and/or core antigens, an antigenfrom hepatitis C virus, Bordetella pertussis, such as pertussisholotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis,optionally also in combination with pertactin and/or agglutinogens 2, adiphtheria antigen, such as a diphtheria toxoid, a tetanus antigen, suchas a tetanus toxoid, a saccharide antigen from Haemophilus influenzae B,an antigen from N. gonorrhoeae) e.g. International patent applicationpublication WO99/24578; WO99/36544; WO99/57280). Other antigens ofinterest include antigens from: Chlamydia pneumoniae (e.g. Internationalpatent application WO0202606; International patent applicationpublications: WO99/27105; WO00/27994; WO00/37494), Chlamydia trachomatis(e.g. International patent application WO99/28475), Porphyromonasgingivalis, polio antigen(s) such as IPV or OPV, rabies antigen(s) suchas lyophilised inactivated virus (e.g. 77, RabAvert™), measles, mumpsand/or rubella antigens, influenza antigen(s), such as thehaemagglutinin and/or neuraminidase surface proteins, the Respiratorysyncytial virus, e.g. the F or the G proteins, the caliciviridae familyof viruses, e.g. norovirus and sapovirus, the reoviridae family, e.g.Rotavirus, herpes simplex viruses, prions, the Salmonella bacteria,Escherichia coli bacteria, the Vibrio cholera bacteria, Moraxellacatarrhalis, Streptococcus agalactiae (group B streptococcus) [e.g.International patent application PCT/GB01/04789], Streptococcus pyogenes(group A streptococcus) [e.g. International patent applicationPCT/GB01/04789], Staphlylococcus aureus, the Respiratory syncytialvirus, e.g. the F or the G proteins, the caliciviridae family ofviruses, e.g. norovirus and sapovirus, the reoviridae family, e.g.Rotavirus, herpes simplex viruses, Salmonella bacteria.

A saccharide or carbohydrate antigen may be conjugated to a carrierprotein Exemplary carrier proteins are bacterial toxins or toxoids, suchas diphtheria, cholera, E. coli heat labile or tetanus toxoids,CRM.sub.197 diphtheria toxoid, N. meninigitidis outer membrane protein[European patent application 0372501], synthetic peptides [Europeanpatent applications 0378881 & 0427347], heat shock proteins[International patent application WO93/17712], pertussis proteins[International patent application WO98/58668; see also EP 04711771,protein D from H. influenzae [International patent applicationWO00/56360], toxin A or B from C. difficile [International patentapplication WO00/61761], for example. Any suitable conjugation reactioncan be used, with any suitable linker where necessary.

Nucleic Acid Encoding Antigen.

The immunomodulatory/adjuvant compositions disclosed may include anucleic acid encoding antigen encoding a polypeptide antigen or aprotein antigen as described above. Examples of antigens that can beencoded by nucleic acids and provided as DNA or RNA-based vaccines andvector vaccines include vaccines for HIV, herpes, hepatitis andinfluenza.

Examples of Compositions

Exemplary compositions are provided in Table 1 below.

TABLE 1 Pharmaceutically Flavonoid (or Vitamin (or Adjuvant AcceptableOil derivative or salt derivative or salt Composition Carrier thereof)thereof) 1 Mustard Oil (+/−AIT) Catechin hydrate Vitamin A 2 Mustard Oil(+/−AIT) Catechin hydrate Vitamin E 3 Mustard Oil (+/−AIT) Catechinhydrate Vitamin C 4 Mustard Oil (+/−AIT) Catechin hydrate Vitamin D 5Mustard Oil (+/−AIT) Catechin hydrate Vitamin A + Vitamin E 6 MustardOil (+/−AIT) Catechin hydrate Vitamin A + Vitamin D 7 Mustard Oil(+/−AIT) Catechin hydrate — 8 Mustard Oil (+/−AIT) — Vitamin A 9 MustardOil (+/−AIT) — Vitamin E 10 Mustard Oil (+/−AIT) — Vitamin C 11 MustardOil (+/−AIT) — Vitamin D 12 Mustard Oil (+/−AIT) — Vitamin A + Vitamin E13 Mustard Oil (+/−AIT) — Vitamin A + Vitamin D 14 Olive Oil (+/−AIT)Catechin hydrate Vitamin A 15 Olive Oil (+/−AIT) Catechin hydrateVitamin E 16 Olive Oil (+/−AIT) Catechin hydrate Vitamin C 17 Olive Oil(+/−AIT) Catechin hydrate Vitamin D 18 Olive Oil (+/−AIT) Catechinhydrate Vitamin A + Vitamin E 19 Olive Oil (+/−AIT) Catechin hydrate —20 Olive Oil (+/−AIT) Catechin hydrate Vitamin A + Vitamin D 21 OliveOil (+/−AIT) — Vitamin A 22 Olive Oil (+/−AIT) — Vitamin E 23 Olive Oil(+/−AIT) — Vitamin C 24 Olive Oil (+/−AIT) — Vitamin D 25 Olive Oil(+/−AIT) — Vitamin A + Vitamin E 26 Olive Oil (+/−AIT) — Vitamin A +Vitamin E

The adjuvant compositions 1-26 described above are exemplary and mayinclude additional components, such as, an additional oil carrier, e.g.,sunflower seed oil, coconut oil, soybean oil. In other embodiments,compositions 1-26 described above do not contain additional oilcarriers, e.g., sunflower oil, coconut oil, soybean oil.

The adjuvant compositions 1-26 described in Table 1 as well as otheradjuvant compositions described in the specification may includeadditional components, such as, additives, e.g., antigens,preservatives, colorants, flavorants, buffers, salts, etc.

Components of the Adjuvant Compositions and their Relative Amounts

The adjuvant compositions described herein may be used to induce animmune response, such as, a Th-1 response. Th-1 response may beparticularly suited to respond to viral infections, intracellularpathogens, and tumor cells because it includes IL-2 and IFN-γ, whichactivate CTLs.

The adjuvant compositions described herein may be used to induce animmune response, such as, a Th-2 response. Th-2 response may be moresuited to respond to extracellular bacteria and helminthic parasites andmay also mediate allergic reactions, since IL-4 and IL-5 are known toinduce IgE production and eosinophil activation.

Vitamin A (e.g., All-trans retinoic acid (ATRA), an acid derivation ofVitamin A) may be included in the adjuvant compositions if a higher Th-2response is desired. In certain cases, it may be desirable to elicit aTh-1 type immune response. In these cases, the adjuvant compositions mayinclude Vitamin C (e.g., ascorbate) and/or Vitamin D and/or Vitamin Eand/or a flavonoid.

The adjuvant composition may include: a flavonoid and Vitamin A; or aflavonoid, Vitamin C and Vitamin A; or a flavonoid, Vitamin D andVitamin A; or a flavonoid, Vitamin E and Vitamin A; or a flavonoid,Vitamin C and E and Vitamin A; or a flavonoid, Vitamin C and D andVitamin A; or a flavonoid, Vitamin D and E and Vitamin A in the adjuvantcompositions. It is understood that each of the foregoing adjuvantcompositions include a pharmaceutically acceptable vegetable oil (e.g.,mustard oil, corn oil, soybean oil, sunflower oil, etc). In addition, itis understood that each of the foregoing compositions may include thevitamin named or a salt or derivative thereof. Similarly, the flavonoidmay be a flavonoid or a salt or derivative thereof.

In certain embodiments, the inclusion of a pharmaceutically acceptablevegetable oil (e.g., mustard oil), a flavonoid, and a vitamin (such as,Vitamin A, C, D and/or E) in the adjuvant compositions may produce anenhanced immune response (for example, synergistic effect) compared tothe effect of an adjuvant composition that includes a pharmaceuticallyacceptable vegetable oil (e.g., mustard oil) and a flavonoid or apharmaceutically acceptable vegetable oil (e.g., mustard oil) and avitamin (such as, Vitamin A, C, D and/or E).

The adjuvant compositions disclosed herein may include mustard oil oranother pharmaceutically acceptable oil carrier. Pharmaceuticallyacceptable oil carrier with rubefacient properties, for example mustardoil, is suitable for preparation of adjuvant compositions foradministration through epithelial cells of the mucosal membranes or theskin or directly injected by e.g. intra-muscular or intra-dermaladministrations.

The volume of pharmaceutically acceptable oil carrier, e.g., mustardoil, used in liquid form in the adjuvant compositions described hereinmay be in the range of 1-95% of the total volume of an adjuvantcomposition. Thus, in certain cases, the pharmaceutically acceptable oilcarrier may make up 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95% by volume of the total volume of a subject adjuvant composition.

The amount of a vitamin (or its salt or derivative) that may be includedin the subject adjuvant compositions may be determined based on the bodyweight of the subject. In general, the recommended daily allowance maybe used to ascertain the amount of vitamin that may be present in thesubject adjuvant compositions.

For example, the amount of vitamin A that may be included in the subjectadjuvant compositions may be in the range of 1-250 μg/kg body weight,e.g., 1 μg/kg, 5 μg/kg, 10 μg/kg, 20 μg/kg, 30 μg/kg, 50 μg/kg, 70μg/kg, 90 μg/kg, 110 μg/kg, 130 μg/kg, 150 μg/kg, 170 μg/kg, 190 μg/kg,210 μg/kg, 230 μg/kg, or 250 μg/kg body weight.

For example, the amount of vitamin C that may be included in the subjectadjuvant compositions may be in the range of 1-100 mg/kg body weight,e.g., 1, 5, 10, 15, 30, 50, 70, 80, 90, or 100 mg/kg body weight.

For example, the amount of vitamin D that may be included in the subjectadjuvant compositions may be in the range of 0.01-10 μg/kg body weight,e.g., 0.01, 0.5, 1, 2, 5, 7, 8, 9, or 10 μg/kg body weight.

For example, the amount of vitamin E that may be included in the subjectadjuvant compositions may be in the range of 0.01-10 μg/kg body weight,e.g., 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 7, 8, 9, or 10 μg/kg body weight.

The amount of a flavonoid (or its salt or derivative) that may beincluded in the subject adjuvant compositions may be determined based onthe body weight of the subject. The amount of the flavonoid, e.g.,catechin (such as catechin hydrate), may be in the range of 1-100 mg/kgbody weight of a subject, e.g., 1, 5, 10, 15, 30, 50, 70, 80, 90, or 100mg/kg body weight.

The adjuvant compositions may be in the form of a suspension, tablet (tobe swallowed or chewed), fast-dissolving tablets or gels or strips,capsule, powder, gel, cream, lotion, ointment, aerosol or the like.

An exemplary adjuvant composition for administration to a human subjectmay include 3-80% vol/vol of mustard oil, e.g., 3%, 10%, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, or 80% vol/vol of mustard oil.

An exemplary adjuvant composition for administration to a human subjectmay include 0.1-100 mg of Vitamin A, e.g., 0.1, 0.5, 1, 5, 10, 15, 30,50, 70, 80, 90, or 100 mg of Vitamin A.

An exemplary adjuvant composition for administration to a human subjectmay include 0.1-400 mg of Vitamin E, e.g., 0.1, 0.5, 1, 5, 10, 15, 30,50, 70, 80, 90, 100, 200, 300, or 400 mg of Vitamin E.

An exemplary adjuvant composition for administration to a human subjectmay include 0.1-2000 mg of Vitamin C, e.g., 0.1, 0.5, 1, 10, 30, 100,130, 200, 300, 600, 900, 1000, 1300, 1500, 1800, or 2000 mg of VitaminC.

An exemplary adjuvant composition for administration to a human subjectmay include 0.1-2000 mg of Catechin hydrate, e.g., 0.1, 0.5, 1, 10, 30,100, 130, 200, 300, 600, 900, 1000, 1300, 1500, 1800, or 2000 mg ofCatechin hydrate.

An exemplary adjuvant composition for administration to a human subjectmay include 0.001-10 mg of AIT, e.g., 0.001, 0.005, 0.01, 0.05, 0.1,0.5, 1, 2, 5, 7, 8, 9, or 10 mg of AIT.

As used in herein vol/vol refers to the volume of a component in thetotal volume of the adjuvant composition.

Th1-type immune response can be induced in mammals by administration ofcertain immunomodulatory polynucleotides. The immunomodulatorypolynucleotides include sequences referred to as immunostimulatorysequences (“ISS”), often including a CG dinucleotide. See, e.g., PCTPublications WO 98/55495, WO 97/28259, U.S. Pat. Nos. 6,194,388 and6,207,646. Thus, in certain embodiments, the subject adjuvantcompositions may include ISS.

In certain embodiments, the subject adjuvant compositions may include anemulsifier (such as, lecithin, or a surfactant, e.g., detergents) asdescribed above. The concentration of an emulsifier in the adjuvantcomposition is dependent on different factors. For example, the higherthe concentration of the pharmaceutically acceptable oil in the adjuvantcomposition the more emulsifier is required. In general, theconcentration of a surfactant or other emulsifier in the subjectadjuvant composition is from 1.5% to 5% v/v, or 1.5% to 3% v/v, or 1.5%to 2.5%, or 2% v/v. When more than one surfactant is used, the sum ofthe concentrations of all surfactants used in the adjuvant compositionis also from 1.5% to 5%, or 1.5% to 3%, or 1.5% to 2.5%, or 2% v/v.

The adjuvant compositions disclosed herein may not include an antigen.An adjuvant composition that does not include an antigen may be used togenerally and non-specifically enhance immune responses, for example toserve as a general immunopotentiator to be taken daily. Alternatively,an adjuvant composition that does not include an antigen can beadministered in conjunction with an antigen, i.e., before,simultaneously, or after vaccinations.

Adjuvant Compositions Including Antigen

In certain embodiments, the subject adjuvant compositions may includeone or more antigens.

The concentration of antigen in adjuvant compositions can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the subject's needs. The concentration of anantigen in the pharmaceutical formulations can vary widely, i.e., fromless than about 0.1%, usually at or at least about 2% to as much as 20%to 30% or more by weight/volume, and will be selected primarily bynature of the antigen, fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. In certainembodiments, the antigen may be 0.1%-30% w/v of the adjuvantcomposition, for example, 0.1%-25%, 0.5%-20%, 1%-15%, 2%-10%, 3%-8%, or5%-6% w/v of the adjuvant composition.

Method of Making Adjuvant Composition

The components of the subject adjuvant composition may be obtained froma variety of sources using a number of methods. Alternatively, thecomponents may be synthesized chemically. In certain cases, thecomponents may be made isolated from a natural source and may beadditionally modified, e.g., chemically modified. For example, mustardoil may be extracted from mustard plant seeds. Alternatively, thepharmaceutically acceptable vegetable oil carrier may be purchased froma vendor. Vitamins A, C, D, and E may be purchased from Sigma Aldrichchemical company, prepared and produced by standard biochemical methods.The flavonoids, e.g., catechins, for example, catechin hydrate, may bepurchased from Sigma Aldrich chemical company, prepared and produced bystandard biochemical methods.

In general, Catechins may either be extracted from green tea orsynthesized chemically. Korean and Chinese green tea, and pu-erh, Indianblack, Longjing, Tieguanyin, Bamboo, Jasmine, Oolong, Flower, Red teasmay be used for extracting catechins, such as, epigallocatechin,catechin, epicatechin, epigallocatechin gallate and epicatechin gallate.Chinese green tea is a rich source of catechin. Green tea is a bettersource of catechin compared to the other types of tea.

Vitamin A (e.g., retinoic acid), Vitamin D (e.g., Calcitriol(1,25-Dihydroxycholecalciferol), Vitamin E (e.g., alpha-tocopherol) andcatechin hydrate may be dissolved in ethanol, for example, 200 proofethanol. Vitamin C may be dissolved in an alkaline solution such assodium bicarbonate buffer. An antigen may be dissolved in water, abuffer (e.g., PBS), or saline solution. A stock solution of theindividual components of the adjuvant composition may be made and theappropriate volumes of the components may then be mixed together toobtain the subject adjuvant composition. The total volume of the subjectadjuvant composition may be adjusted with PBS or saline.

In certain embodiments, a pharmaceutically acceptable oil and aflavonoid and optionally a vitamin may be mixed together in amounts asdescribed above along with a surfactant such as Tween®-80. Beforeadministrating, the adjuvant composition may be emulsified by repeatedlywithdrawing and releasing the mixture of a pharmaceutically acceptableoil, a surfactant(s), and another component(s).

The components of the adjuvant compositions may be sterilized prior toadmixing or after forming the adjuvant compositions. The adjuvantcompositions may be mixed with a gel, or formulated into microparticles,etc. before administration.

The adjuvant compositions disclosed herein may be formulated into aspray (e.g., nasal spray), drops (e.g., nasal drops), gel, powder,tablets or capsules, patch, and the like. Of particular interest areadjuvant compositions suitable for administration via inhalationincluding but not limited to, liquid suspensions for forming aerosols aswell as powder forms for dry powder inhalation delivery systems. Devicessuitable for administration by inhalation of subject adjuvantcomposition include, but are not limited to, atomizers, vaporizers,nebulizers, and dry powder inhalation delivery devices.

The adjuvant compositions disclosed herein may be formulated intoliquids or emulsions for injecting using a parenteral route, e.g.,intravenous, intramuscular, subcutaneous.

Method of Using Adjuvant Compositions

The present disclosure provides methods for modulating an immuneresponse in a subject, such as, stimulating a cellular and/or a humarlimmune response. The adjuvant compositions disclosed herein can beuseful for prophylaxis and/or treatment of various infections andneoplastic diseases.

Conditions

In certain embodiments, the adjuvant compositions disclosed herein maybe find use in the context of administering an antigen, as a vaccine.The vaccine may be a prophylactic vaccine or a therapeutic vaccine. Aprophylactic vaccine is given before infection to prevent disease,whereas a therapeutic vaccine is give after the onset of infection ordisease. A prophylactic vaccine comprises one or more epitopesassociated with a disorder for which the subject may be at risk (e.g.,M. tuberculosis antigens as a vaccine for prevention of tuberculosis).Therapeutic vaccines comprise one or more epitopes associated with aparticular disorder affecting the individual, such as M. tuberculosis orM. bovis surface antigens in tuberculosis patients, antigens to whichthe individual is allergic (i.e., allergy desensitization therapy) inindividuals subject to allergies, tumor cells from an individual withcancer (e.g., as described in U.S. Pat. No. 5,484,596), or tumorassociated antigens in cancer patients.

The adjuvant composition may be given in conjunction with the antigen(e.g., in the same composition or a simultaneously using separatecompositions) or the adjuvant composition may be administered separately(e.g., at least 12 hours before or after administration of the antigen).In certain embodiments, the antigen(s) is admixed with the adjuvantcomposition.

Administration of the subject adjuvant composition and antigen mayresult in amelioration of one or more symptoms or a later onset of oneor more symptoms of the disorder which the vaccine is intended to treat.As will be apparent to one of skill in the art, the exact symptoms andmanner of their improvement will depend on the disorder sought to betreated. For example, where the therapeutic vaccine is for tuberculosis,administration of adjuvant composition with antigen may result inreduced or delayed onset of coughing, pleural or chest wall pain, fever,and/or other symptoms known in the art. Where the vaccine is an allergenused in allergy desensitization therapy, the administration of adjuvantcomposition with antigen may result in a reduction or a delay in onsetof the symptoms of allergy (e.g., reduction in rhinitis, allergicconjunctivitis, circulating levels of IgE, and/or circulating levels ofhistamine).

The subject adjuvant composition may also be used prophylactically toincrease resistance to infection by a wide range of bacterial or viralpathogens, including natural or genetically modified organisms employedas agents of biological warfare or bio-terrorism.

Other embodiments relate to immunomodulatory therapy of subjects havinga pre-existing disease or disorder, such as cancer or an infectiousdisease. Cancer is an attractive target for immunomodulation becausemost cancers express tumor-associated and/or tumor specific antigenswhich are not found on other cells in the body. Stimulation of aTh1-type immune response results in the death of tumor cells, either bydirect action of cellular immune system cells (e.g., CTLs) or componentsof the humoral immune system, or by bystander effects on cells proximalto cells targeted by the immune system including macrophages and naturalkiller (NK) cells.

The adjuvant composition disclosed herein can also be administered tosubjects with infectious diseases caused by extracellular pathogens(e.g., bacteria or protozoans) or by intracellular pathogens (e.g.,viruses).

In certain embodiment, a subject suffering from a disorder associatedwith a Th2-type immune response, such as (without limitation) allergies,allergy-induced asthma, atopic dermatitis, eosinophilic gastrointestinalinflammation, eosinophilic esophagitis, and allergic bronchopulmonaryaspergillosis may be treated by administering an adjuvant compositiondisclosed herein. For example, an adjuvant composition comprising apharmaceutically acceptable oil carrier, a flavonoid and at least onevitamin C, D and E may be administered to the subject suffering from adisorder associated with a Th2-type immune response increasing levels ofone or more Th1-type response associated cytokines, which may result ina reduction of the Th2-type response features associated with thesubject's response to the allergen. Immunomodulation of a subject withTh2-type response associated disorders results in a reduction orimprovement or delay in the onset of one or more of the symptoms of thedisorder. Where the disorder is allergy or allergy-induced asthma,improvement in one or more of the symptoms includes a reduction one ormore of the following: rhinitis, allergic conjunctivitis, circulatinglevels of IgE, circulating levels of histamine and/or requirement for“rescue” inhaler therapy (e.g., inhaled albuterol administered bymetered dose inhaler or nebulizer).

Route of Administration

The adjuvant compositions disclosed herein may be administered to asubject via a number of routes of administration. Exemplary routes ofadministration include mucosal, e.g., oral, sublingual, intra-nasal,inhalation, ocular, intra-vaginal, intra-rectal; and systemic, e.g.,intra-muscular, intra-dermal, trans-dermal, intraperitoneal,subcutaneous or trans-cutaneous. In certain embodiments, a combinationof at least two routes of administration may be used to induce an immuneresponse. For example, a combination of a mucosal route and a systemicroute of administration may be used.

In certain embodiments, the adjuvant compositions described herein arenot administered systemically.

The route of administration may vary based on the individual subject andthe stage of the disease and other factors evident to one skilled in theart.

In certain embodiments, the adjuvant compositions described herein maybe administered through the mucosal surface without breaking the mucosalsurface.

The subject adjuvant compositions may be used with or without anantigen(s). When used with an antigen, the adjuvant composition and theantigen may be administered simultaneously or the adjuvant compositionmay be administered before or after the administration of the antigen.When used with an antigen, the antigen may be mixed with the adjuvantcomposition.

The adjuvant compositions disclosed herein may be provided as micro- ornano-particles in gel or tablet (such as, fast dissolving) forms. Suchformulations may be administered via oral or sublingual routes, forexample. For intra-nasal administration, the adjuvant compositions maybe formulated as nasal sprays in an emulsion form or drops, for example.For transcutaneous administration, adjuvant compositions may be given ina gel, lotion or ointment form. For systemic injections, the adjuvantcompositions can be given formulated as an emulsion and/ormicro/nanoparticles. For rectal administration, the adjuvantcompositions can be formulated as suppository or gels, for example. Forvaginal administration, the adjuvant compositions formulated as gel,emulsion, ointment, for example.

In certain embodiments, the adjuvant compositions disclosed herein maybe administered to a subject via a combination of different routes inthe order indicated below:

i. systemic, mucosal;ii. systemic, systemic, mucosal, mucosal;iii. systemic, mucosal, systemic;iv. mucosal, mucosal, systemic, systemic;v. mucosal, systemic, systemic;vi. mucosal, systemic, mucosal, for example.

When an adjuvant composition is administered systemically or mucosallymore than once, the two or more systemic or mucosal administrations maybe by the same systemic (for example, two intramuscular injections) ormucosal route (two IN/SL administrations) or different (for example, oneintramuscular injection and one intravenous injection; one INadministration and one SL administration).

Dosages

The dosage of the adjuvant compositions described herein to beadministered to a subject comprising may be determined based on theroute of administration and body weight and may range from 0.001 ml/kgbody weight to 1 ml/kg body weight. The number of times an adjuvantcomposition is administered may vary and may be determined based uponnumerous factors. These factors are evident to a person of skill in theart and may include, the disease to be prevented or treated, the type ofpathogen or cancer, the structural nature of the antigen, the route ofadministration, the level of immune response induced in the subject, thetype of immune response, etc.

Subjects

The adjuvant compositions described herein may be used to elicit animmune response in a variety of subjects capable of mounting an immuneresponse. In certain cases, the adjuvant compositions described hereinmay be administered to any member of the subphylum chordata, including,mammals (humans, other non-human primates, domesticated animals, e.g.,livestock), avians, fishes, or any other animal in need thereof. Incertain cases, the adjuvant compositions may be administered to humans.In certain cases, the adjuvant compositions may be administered to cows.In certain cases, the adjuvant compositions may be administered tochickens, horse, sheep, goats. In certain cases, the adjuvantcompositions may be administered to porcines. In certain cases, theadjuvant compositions may be administered to cats and dogs.

Detection of Immune Response

Modulation of an immune response may be humoral and/or cellular, and maybe measured using standard techniques in the art. An immune response ina subject can be detected in any number of ways, including measuringexpression levels of antigen-specific antibodies, one or more ofIFN-gamma, IFN-alpha, IL-2, IL-12, TNF-alpha, IL-6, IL-4, IL-5, IL-10,IL-12, IL-13, IL-15, IL-18, IL-22, and other cytokines as well asdetecting responses such as B cell proliferation, activation of specificpopulations of lymphocytes such as CD4⁺T cells, NK cells or CTLs, anddendritic cell and macrophage maturation and activation.

Methods for measuring specific antibody responses include enzyme-linkedimmunosorbent assay (ELISA) and are well known in the art. Measurementof numbers of specific types of lymphocytes such as CD4⁺T cells can beachieved, for example, with fluorescence-activated cell sorting (FACS).Cytotoxicity and CTL assays can be as described in Raz et al. (1994)Proc. Natl. Acad. Sci. USA 91:9519-9523, for example. Cytokineconcentrations can be measured, for example, by ELISA. These and otherassays to evaluate the immune response to an immunogen are well known inthe art. See, for example, SELECTED METHODS IN CELLULAR IMMUNOLOGY(1980) Mishell and Shiigi, eds., W. H. Freeman and Co.

Kits

Kits that include one or more sterile containers of components of theadjuvant compositions described herein are also provided. Individualcomponents may be present in separate sterile containers or two or morecomponents may be present in a single container. Optionally, the kit mayalso include a container containing a desired antigen(s).

In some embodiments, the sterile containers may optionally have anaccess port(s) for withdrawing a specific volume/amount of a component,for example, a port for introducing a syringe to withdraw a certainvolume of a pharmaceutically acceptable oil.

In some embodiments, the containers of the components of the adjuvantcompositions described herein may not be sterile but are reasonablyclean.

The kits may further include a suitable set of instructions, generallywritten instructions, relating to the use of the adjuvant compositionfor immunomodulation (e.g., ameliorating symptoms of an infectiousdisease, increasing IFN-gamma levels, increasing IFN-alpha levels, orameliorating an IgE-related disorder).

The kits may comprise the components of the adjuvant compositionpackaged in any convenient, appropriate packaging. For example, if acomponent is a dry formulation (e.g., freeze dried or a dry powder), avial with a resilient stopper may be used, so that the component may beeasily resuspended by injecting fluid through the resilient stopper.Ampoules with non-resilient, removable closures (e.g., sealed glass) orresilient stoppers may be used for liquid component(s) of the adjuvantcomposition. Also contemplated are packages for use in combination witha specific device, mucosal administration devices, such as, an inhaler,nasal administration device (e.g., an atomizer) or eye drops.

The instructions relating to the use of adjuvant composition generallyinclude information as to dosage, dosing schedule, and route ofadministration for immunomodulation. The containers of containing thecomponents of adjuvant composition or the premixed adjuvant compositionmay be unit doses, bulk packages (e.g., multi-dose packages) or sub-unitdoses. Instructions supplied in the kits disclosed herein are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) may also beincluded.

EXAMPLES

The following example is provided to further illustrate the advantagesand features of the present invention, but is not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Materials and Methods

The following materials and methods were used in Examples 1-4.

Adjuvants and Vaccine Preparations.

Recombinant HA protein of A/Calcdonia/20/99 (H1N1)-like virus (stockconcentration 0.33 μg/μl) was used at 0.5 μg per dose. Vitamin A (VA;Retinoic acid, Sigma-Aldrich; Cat# R2625), Vitamin E (VE; alphatocopherol, Sigma-Aldrich/Fluka Biochemika; Cat#95240) and CatechinHydrate (Cat; Sigma-Aldrich, C1251, synonym: (+)-Cyanidol-3,(2R,3S)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol)were dissolved in 200 proof absolute ethanol (Sigma-Aldrich,Cat#459844). The stock concentration was 3 μg/μl for VA, 2000 μg/μl forVE, and 50 μg/μl for Cat Hydrate. Vitamin C (VC; Sigma-Aldrich, Cat#A4544) was dissolved in Sodium Bicarbonate buffer (pH 10.6) at theconcentration of 100 μg/μl (pH 5.5). Mustard seed oil (MO) was purchasedfrom Botanic Oil Innovations Inc. (Spooner, Wis.).

The amount used for each dose of vaccination was 30 μg for VA, 500 μgfor VC, 2000 μg for VE, and 120 μg for Cat. The total volume for each IMthigh dose was 100 μl which was adjusted with Dulbecco's phosphatebuffered saline (PBS) (Cat#21-030-CV). All vaccines were prepared withendotoxin free reagents and in endotoxin free 2.0 ml tubes (Eppendorfbiopur safe-lock microcentrifuge tubes).

A total of 22 different adjuvant combinations with vaccine wereprepared. The first 11 groups were prepared without MO, and the last 11groups were prepared with MO (50% of the total volume). For allcombinations with MO, Tween®-20 (Sigma, Cat# P1379) was added at a finalconcentration of 0.1% for emulsification. The combinations with MO wereemulsified by repeatedly withdrawing-releasing for at least 15 timesusing BD's 1/2 CC ½ In. 27 G tuberculin syringes (Cat#305620). Fourdoses were prepared for each combination.

The 22 groups of combinations of HA vaccines were as shown in Table 2below. Each group includes recombinant HA protein.

TABLE 2 GROUP VACCINE COMPOSITION 1 VA 2 VC 3 VE 4 Cat 5 VA + VC 6 VC +VE 7 VC + Cat 8 VE + Cat 9 VC + VE + Cat 10 VA + VC + VE + Cat 11 PBS 12VA + MO 13 VC + MO 14 VE + MO 15 Cat + MO 16 VA + VC + MO 17 VC + VE +MO 18 VC + Cat + MO 19 VE + Cat + MO 20 VC + VE + Cat + MO 21 VA + VC +VE + Cat + MO 22 MO

Animals and Immunizations.

Sixty-six female BALB/c mice of 6-8 weeks of age were randomly dividedinto 22 groups (3 mice/group). The mice were maintained according toIACUC guidelines in the vivarium of Murigenics Inc. (Vallejo, Calif.94592). The mice were anesthetized with isoflurane, and vaccinated byintramuscular (IM) injections at both thighs using the same tuberculinsyringes used for emulsification. Each mouse received 100 μl of thevaccine preparations, i.e., 50 μl at each thigh.

Test of Cytokines in Sera Collected One Day after Immunization.

At one day post vaccination, mice were bled retro-orbitally and serumwas prepared from individual mice. The sera were tested for TNFα andIL-12 using ELISA Max™ Deluxe sets from Biolegend (Cat#433606 andCat#430906, respectively) following the manufacturer's instructions. Toenhance the sensitivity of the tests, an ELAST® ELISA AmplificationSystem by PerkinElmer (Cat#NEP116E001EA) was used. Briefly, after theavidin-HRP step in the Biolegend kits, biotinylated tyramide (10 μl/mlin tyramide diluent) was added. The plates were incubated for 15 minutesat room temperature. After washing with PBS-Tween20 (PBS-T) 5 times,streptavidin-HRP was added at 2 μl/ml in blocking solution (1% goatserum and 0.02% Tween-20 in PBS). After incubation for 30 minutes atroom temperature, the plates were washed 5 times with PBS-T and thesubstrate TMB (BioFX Laboratories, Cat# TMBS-0100-01) was added. Thecolor development was watched closely and stopped immediately with 1NH₂SO₄ when background color started to appear in blank wells. Absorbancewas read at 450 nm using a Molecular Devices UV max kinetic microplatereader (Sunnyvale, Calif.). The concentration of each cytokine in eachsample was determined using the Softmax Pro software (Molecular Devices,Sunnyvale, Calif.) and based on standards provided by the kitmanufacturer.

Test of Serum Antibodies by ELISA.

At 3 weeks post vaccination, the mice were bled retro-orbitally. Serawere collected for individual mouse and were kept at −20° C. beforetests. For IgG1 ELISA, nunc Maxisorp® 96-well microplates were coatedwith 100 μl of the recombinant HA protein at the concentration of 0.66μg/ml in PBS at 4° C. overnight. After washing 4 times with PBS-T, theplates were blocked with 200 μl blocking solution (1% goat serum, 0.02%Tween-20 in PBS) for 1 hour at room temperature. The serum samples werediluted 1/600 in the blocking solution initially and 3-fold serialdilutions were made in each column of 96-well microplates for eachsample. Column 1 was used as blank. A positive reference serum and anegative reference serum were diluted in the same way in column 2 and 3,respectively. After addition and dilution of the serum samples, theplates were incubated for 2 hours at room temperature. The plates werethen washed 5 times with PBS-T followed by addition of 100 μl goatanti-mouse IgG1-HRP conjugate (Southern Biotech, Cat#1070-05) diluted1/8000 in blocking solution. The plates were incubated for 1 hour atroom temperature. After washing 5 times with PBS-T, 100 μl TMB substratewas added for color development. The color development was stopped at 30minutes by addition of 100 μl of 1N sulfuric acid. The plates were readat 450 nm using a microplate reader (Molecular Devices, Sunnyvale,Calif.). The cutoff value was determined as two and half times of thebackground value (average value of blank wells). The titer of each serumsample was calculated automatically using the software based onlog-logit curves. For IgG2a titration, all the procedures and reagentswere the same as for IgG1 ELISA except for the secondary antibodyconjugate. A goat anti-mouse IgG2a-HRP conjugate (Southern Biotech,Cat#1080-05) diluted at 1/8000 was used. For IgA titration, theprocedures and reagents used were the same as above except that theserum samples were diluted initially at 1/20 followed by 3-fold serialdilutions in each column and a goat anti-mouse IgA-Biotin (SouthernBiotech, Cat#0106-08) diluted 1/8000 was used. An additional step withavidin-HRP was also needed.

Splenocyte Cultures and Tests of Cytokines in Supernatants by ELISA.

One week post 2^(nd) vaccination the 66 mice were sacrificed, andspleens (SP) and vaginal lavages were collected. Each individual spleenwas pressed through Falcon 70 um nylon mesh (Cat#353910) followed byrinsing of the mesh with about 4.5 ml complete RPMI 1640 media with 5%FBS and antibiotics (complete RPMI). The cells were collected into a 50ml centrifuge tube and were transferred into a 5 ml Nalgene tube(Cat#5000-0050). After spinning for 5 minutes at 4° C. at 600×g in aEppendorf Benchtop centrifuge (5702R), the cells were gently resuspendedin 4.5 ml complete RPMI. The cells were centrifuged and resuspendedagain. For cell counting, 20 μl cell suspensions were mixed with 180 ulPBS to make a 1/10 dilution, and 10 μl of the diluted cell suspensionwas mixed with 10 μl trypan blue, and 10 μl of the mixture was used forcounting the cells with a hemocytometer. After counting, the cellconcentration in each spleen sample was diluted to a final concentrationof 2×10⁶/ml.

For cell culture, Corning 24-well cell culture plates (Cat#3526) wereused. 0.5 ml of the cells was added to each well (1×10⁶ cells/ml). Twowells were used for each sample. One well contained 0.5 ml complete RPMI1640 and the other well contained 0.5 ml complete RPMI 16490 with 2μg/ml HA protein resulting in the final concentration of 1 μg/ml. Afterincubation at 37° C. in a CO₂ incubator for 24 hours, cells andsupernatants (SN) were harvested and were centrifuged for 5 minutes at600×g. The supernatants were collected in 2 ml microcentrifuge tubes andstored at −20° C. and assayed within a week.

To test cytokines in the supernatants, IFNγ, IL-5, IL-10 were testedusing ELISA Max™ Deluxe sets from Biolegend (Cat#430806, Cat#431206, andCat#431406, respectively) following the manufacturer's instructions. Toenhance the sensitivity of the tests, the ELAST® ELISA AmplificationSystem by PerkinElmer was used and the procedure was the same describedabove.

Test of IgG1 and IgA in Vaginal Lavages by ELISA.

Vaginal washes about 100 μl were collected from individual mouse using apipetman by aspirating about 100 μl PBS inside the vaginal vault 5-7times. The vaginal lavages were kept on dry ice for later use. Spleenswere removed aseptically from individual mice following the collectionof vaginal lavages and were immediately placed into 4.5 ml cold RPMI1640 basic medium (UCSF Cell culture facility). Vaginal washes collectedabove were thawed and vortexed followed by centrifugation at 3000×g for5 minutes. The 3 vaginal lavages within each group were pooled and testswere run for individual group. The ELISA tests were performed in asimilar way to that described for serum IgG1 and IgA above except thatthe initial dilution for both IgG1 and IgA was 1/3 followed by 3-foldserial dilutions. The tests also employed the PerkinElmer ELAST® ELISAAmplification System described above to increase sensitivity. For IgG1,the biotinylated tyramide was added after the Goat anti mouse IgG1-HRPstep. For IgA, the biotinylated tyramide was added after the avidin-HRPstep.

Example 1 Immune-Modulation Following a Single Intramuscular (IM)Injection

A significant enhancement of serum IgG1 antibody responses followingvaccinations with HA plus selected components was observed. While HAplus MO enhanced the responses compared to HA alone, there was a furtherenhancement with other components. In general, addition of MO enhancedthe IgG1 responses compared to single vitamins, cat or cat+VE (FIG. 1).The enhancement by MO compared to vaccinations with HA in PBS alone, wasthe highest in the group vaccinated with HA+cat+MO, which induced a 15fold enhancement, followed by HA+cat+VE+MO and HA+VA+MO (FIG. 1). Thegroups of mice vaccinated with the other compositions listed in Table 1did not show enhanced serum IgG1 antibody responses compared to HA inPBS alone. These results demonstrate that of all the 22 compositionstested, cat plus MO mixed with HA induced the highest IgG1 responses.Interestingly, the adjuvant compositions with vitamin C appeared tosuppress immune response. However, it is hypothesized that thesuppression of immune response may be an artifact caused by the highdose of vitamin C administered to the mice or the one pH unit differencebetween adjuvant compositions with vitamin C compared to those withoutvitamin C.

Serum anti-HA IgG2a responses were measured as a potential surrogate forTH1 response. In contrast to the IgG1 responses, the highest serum IgG2aresponses were measured in the group vaccinated with VA alone (FIG. 1).

Example 2 Immune-Modulation Following Two Intramuscular (IM) Injections

Compared to the data obtained at 3 weeks after a single IM vaccination,at one week after the second IM vaccination, serum anti-HA IgG1responses generally increased at least 10 fold (FIG. 2). Similar to theresults after a single IM vaccination, anti-HA IgG1 responses werehigher in the groups that were vaccinated with HA+MO, HA+MO+VA, andHA+MO+Cat, compared to vaccinations with HA in PBS alone. In addition,the group that was vaccinated with HA+MO+VE also showed enhanced IgG1responses compared to HA in PBS alone. Addition of MO to any of thegroups enhanced the IgG1 responses. None of the other compositionslisted in Table 1 resulted in the enhancement of the IgG1 responsescompared to HA in PBS alone (data not shown). These data show thatfollowing two IM vaccinations, compared to vaccinations with HA in PBSalone, serum anti-HA responses were significantly enhanced when MO wasmixed with cat, VA or VE.

After the second IM vaccination with HA+VA+MO the IgG2a responses wereenhanced compared to the IgG2a responses measured after the firstvaccination (FIGS. 1 and 2).

To determine whether vaccinations with the various components resultedin heavy chain isotype switch to IgA, serum anti-HA IgA responses weredetermined. Groups of mice vaccinated with HA+VA+MO, HA+VC+MO,HA+Cat+MO, and HA+VE+cat showed an enhancement of serum IgA responsecompared to HA in PBS alone (FIG. 3).

None of other compositions listed in Table 1 resulted in enhanced serumIgA responses.

These data indicate the utility of VA+MO and Cat+MO as adjuvants thatinduce antigen-specific isotype switch to IgA and IgG, respectively.

Example 3 Mucosal Immune Response Following Intramuscular (IM)Injections

Responses Measured in Vaginal Lavages.

The female genital tract is a major mucosal tissue and serves as theroute of transmission of many important bacterial and viral agents.Because induction of antibody responses in this tissue is important forprotection against mucosally transmitted diseases, such as HIV andChlamydia, anti-HA IgG1 and IgA responses in vaginal lavages collectedat 1 week after the second IM vaccinations were measured. The highestvaginal anti-HA IgG1 responses was observed in the mice vaccinated withHA+VA+MO, HA+VE+MO, and HA+VE+cat+MO (FIG. 4). Moreover, the only groupthat showed enhanced anti-HA IgA responses compared to HA in PBS alone,was the group vaccinated with VA+MO (FIG. 5). These data indicate thatcombinations of the MO with VA, VE, cat result in induction of enhancedserum IgG1, IgG2a, as well as vaginal IgG1 and IgA.

Example 4 TH1, TH2, and Treg Responses Following Intramuscular (IM)Injections

TH1, TH2 and Treg Responses after Second IM Vaccination with HA.

TH1 (IFNγ), TH2 (IL-5) and Treg (IL-10) responses in splenocytesprepared at one week after the second IM vaccination and activated exvivo overnight with HA was determined. Enhanced IFNγ responses comparedto that of mice injected with HA in PBS alone were detected in thegroups of mice vaccinated with HA+MO, with further enhancement when MOwas mixed with other components (FIG. 6). The highest amount of IFNγ wasdetected in the group vaccinated with MO+cat (FIG. 6). While the groupsvaccinated with VC+MO, MO alone, VE+cat+MO, and VE±MO showed enhancedIFNγ responses compared to HA alone, the responses were lower than thegroup vaccinated with MO+cat (FIG. 6). These data suggest thatvaccinations with VA and cat+MO induced the highest TH1 responses. Incontrast to TH1 responses, TH2 (IL-5) responses were only enhancedfollowing vaccinations with VA or VE, as the IL-5 responses in thegroups vaccinated with HA alone in PBS were generally high.Interestingly, vaccinations with MO plus VA, VC, VE and VE+cat reducedIL-5 responses, while vaccinations with cat+MO induced higher IL-5responses than vaccinations with cat, but at comparable level asvaccination with HA in PBS alone (FIG. 6). Importantly, the IL-10 Tregsuppressive responses mostly correlated with the poor serum IgG, orsplenic IFNγ or IL-5 responses, in that the highest IL-10 responses weremeasured in the group vaccinated with HA in PBS alone (FIG. 6). Thesedata indicate that IM vaccinations with cat+MO induced the most wellbalanced TH1, and TH2 with a lack of Treg responses.

Example 5 Immune-Modulation Following Intranasal (IN) and Sublingual(SL) Vaccinations

Materials and Methods

Adjuvants and Vaccine Preparations.

The HIV-1 gp120_(cn54) protein was obtained from the NIH AIDS Researchand Reference Reagents Program and was used at the dose of 5 μg permouse. Vitamin A (Retinoic acid, Sigma-Aldrich; Cat# R2625) and CatechinHydrate (Sigma-Aldrich, C1251) were dissolved in 200 proof absoluteethanol (Sigma-Aldrich, Cat#459844). The stock concentration was 3 μg/μlfor Vitamin A (VA), 50 μg/μl for Catechin Hydrate (Cat). Mustard seedoil (MO) was purchased from Botanic Oil Innovations inc. (Spooner,Wis.). Allyl isothiocyanate (AIT) was purchased from Sigma Aldrich (Cat#W203408-500G-K). The amount used for each dose of vaccination was 30 μgfor Vitamin A, 120 μg for Cat, and 1 μl for AIT. The total volume foreach dose was 40 μl and was adjusted with Dulbecco's phosphate bufferedsaline (Cat#21-030-CV). All components used in the vaccine includingvitamin A, Cat, Tween-20, MO, and PBS were tested for endotoxin with aGenscript kit (Piscataway, N.J. 08854; Cat# L00350) and the endotoxincontent in each component was found to be less than 0.005 EU/ml. Allvaccines were prepared in endotoxin free 2.0 ml tubes (Eppendorf biopursafe-lock microcentrifuge tubes). Tween®-20 (Sigma, Cat# P1379) wasadded to all groups at a final concentration of 0.1%. The combinationswith MO were emulsified by repeatedly withdrawing-releasing for at least15 times using BD's 1/2 CC ½ In. 27 G tuberculin syringes (Cat#305620).The volume total dose volume per mouse for each combinedintranasal/sublingual (IN/SL) vaccination was 38.8 μl, i.e. 19.4 μl forIN and 19.4 μl for SL routes. A group of three female BALB/c mice werevaccinated with HIVenvgp120_(cn54)+MO+VA+Cat+AIT (Group 1). Anothergroup of three female BALB/c mice were vaccinated withHIVenvgp120_(cn54) in PBS alone. The mice in both groups were: at 6-8weeks of age. The two groups of mice received the combined IN/SLvaccinations twice at 2 weeks intervals. Serum and vaginal lavages werecollected at 2 weeks after the second vaccination.

The mice were maintained according to IACUC guidelines in the vivariumof Murigenics Inc. (Vallejo, Calif. 94592). Before the firstvaccination, the mice were anesthetized with isoflurane, forsimultaneous intra-nasal (IN) and sublingual (SL) vaccinations, whereasfor the second vaccination the mice were not anesthetized for INadministrations.

Test of Serum Antibodies by ELISA.

The mice were bled retro-orbitally at two weeks after the secondvaccination. Sera were stored at −20° C. To measure anti-HIVgp120_(cn54) IgG1 and IgG2a titers nunc Maxisorp® 96-well microplateswere coated overnight with 2 μg/ml of the HIV gp120_(cn54) protein inPBS (100 μl per well) at 4° C. After washing 4 times with PBS-T (0.02%Tween-20 in PBS), the plates were blocked with 200 μl blocking solution(1% goat serum, 0.02% Tween-20 in PBS) for 1 hour at room temperature.The serum samples were diluted 1/600 in the blocking solution initiallyand 3-fold serial dilutions were made in each column of 96-wellmicroplates for each sample. Column 1 was used as blank. Negativecontrol sera were from naïve mice. After addition and dilution of theserum samples, the plates were incubated for 2 hours at roomtemperature. The plates were then washed 5 times with PBS-T followed byaddition of 100 μl goat anti-mouse IgG1-HRP conjugate (Southern Biotech,Cat#1070-05) diluted 1/8000 in blocking solution. The plates wereincubated for 1 hour at room temperature. After washing 5 times withPBS-T, 100 μl TMB substrate was added for color development. The colordevelopment was stopped in about 15-30 minutes by addition of 100 μl of1N sulfuric acid. The plates were read at 450 nm using a microplatereader (Molecular Devices). The cutoff value was determined as two andhalf times of the background value (average value of blank wells). Thetiter of each serum sample was calculated automatically using thesoftware based on log-logit curve. For IgG2a titration, all theprocedures and reagents were the same as for IgG1 ELISA except for thesecondary antibody conjugate. A goat anti-mouse IgG2a-HRP conjugate(Southern Biotech, Cat#1080-05) diluted 1/8000 was used. For IgAtitration, the procedures and reagents used were the same as aboveexcept that the serum samples were diluted initially at 1/20 followed by3-fold serial dilutions in each column and a goat anti-mouse IgA-Biotin(Southern Biotech, Cat#0106-08) diluted 1/8000 was used. The goatanti-mouse IgA-Biotin was detected using avidin-HRP.

Test of IgG1 and IgA in Vaginal Lavages by ELISA.

Vaginal lavages of about 100 μl were collected from individual mouseusing a pipetman by aspirating about 100 μl PBS inside the vaginaseveral times. The vaginal lavages were kept on dry ice for later use.Vaginal lavages were thawed and vortexed followed by centrifugation at3000×g for 5 minutes. The vaginal lavages were tested for IgG1 and IgAby ELISA. The ELISA tests were performed in a similar way to thatdescribed for serum IgG1 and IgA above except that the initial dilutionfor both IgG1 and IgA was 1/3 followed by 3-fold serial dilutions. Thetests also employed the PerkinElmer ELAST® ELISA Amplification Systemdescribed above to increase sensitivity as per manufacturer's protocols.

Results.

At two weeks following two combined intra-nasal and sublingualvaccinations, serum IgG1 anti-HIVgp120 responses were enhanced anaverage of 198 fold in mice vaccinated with HIVgp120_(cn54) inMO+VA+cat+AIT compared to the mice vaccinated with HIVgp120_(cn54) inPBS alone (FIG. 7). Moreover, serum IgG2a anti-HIVgp120 responses werealso enhanced (FIG. 7). There was also an enhancement of both IgG1 andIgA titers in vaginal lavages in mice vaccinated with HIVgp120_(cn54) inMO+VA+cat+AIT compared to the mice vaccinated with HIVgp120_(cn54) inPBS alone (FIG. 8).

Example 6 Immune-Modulation by IN/SL and IM Vaccinations

Materials and Methods

Groups of 3 female BALB/c mice each at 6-8 weeks of age were vaccinatedwith HIVenvgp120cn54 only in PBS or with HIVenvgp120cn54+MO+VA+Cat+AITtwo times IN/SL route followed by two times IM.

Before the first vaccination, the mice were anesthetized withisoflurane, for simultaneous intra-nasal (IN) and sublingual (SL)vaccinations, whereas for the second vaccination the mice were notanesthetized for IN administrations. The doses of catechin and VA wereas described above for IM vaccinations with HA, i.e., 120 μg and 30 μg,respectively. The dose of HIVgp120 was 5 μg for the IN/SL vaccinationsand the first IM vaccination and 2.5 μg for the second (final) IMvaccination. The dose of AIT was 1 μl. MO was used at 50% v/v for bothIN and IM vaccinations. Thus, the following groups of mice received twocombined IN/SL, followed by two IM vaccinations at 2 weeks intervals:

Group 1: HIVenvgp120cn54+MO+VA+Cat+AITGroup 2: HIVenvgp120cn54 only in PBS

Serum and vaginal lavages were collected at 1 week after the 4th andfinal vaccination, and gp120-specific IgG1, IgG2a and IgA were measuredby ELISA.

Results.

IgG1, IgG2a and IgA responses in serum were markedly enhanced in thegroup of mice vaccinated with HIVenvgp120cn54+MO+VA+Cat+AIT compared tothe group of mice vaccinated with HIVenvgp120cn54 only in PBS (FIG. 9).

IgG1 and IgA responses in vaginal lavages were also next determined.IgG1 and IgA responses in vaginal lavages were markedly enhanced in thegroup of mice vaccinated with HIVenvgp120cn54+MO+VA+Cat+AIT compared tothe group of mice vaccinated with HIVenvgp120cn54 only in PBS (FIG. 10).These data show markedly enhanced serum and vaginal anti-gp120 antibodyresponses and strongly support the immune-enhancing capacity ofMO+VA+Cat+AIT for induction of both vaginal and systemic humoralimmunity against the HIV-1gp120 surface glycoprotein.

Example 7 Adjuvant Composition with Antigen for Administration to aHuman Subject

An example of an adjuvant composition that includes a pharmaceuticallyacceptable vegetable oil carrier, AIT, a flavonoid, and vitamins A, Eand C is provided in Table 3 below:

TABLE 3 Component Amount Mustard Oil 3-80% vol/vol Vitamin A¹ 0.1-100 mgVitamin E² 0.1-100 mg Vitamin C³ 0.1-2000 mg Catechin hydrate⁴ 0.1-2000mg Emulsifier⁵ 0.1-7% w/w Allyl Isothiocyanate 0.001-10 mg

The above composition may include one or more of the components shown inTable 4 below:

TABLE 4 Component Amount Antigen⁶ 0.01-10 mg PBS 1-50% vol/vol Gelatin1-20 mg Antibiotics⁷ 0.1 pg-30 μg Sorbitol 1-20 mg Sucrose⁸ 1-100 mgHyaluronic Acid⁹ 1-30% w/w Beeswax 1-50% w/w ¹Vitamin A may be providedas carotene, beta-carotene, retinoic acid, retinyl palmitate, or aderivate or slat thereof; ²Vitamin E may be provided as alpha-tocopherolor a derivate or slat thereof; ³Vitamin C or a derivate or slat thereof,⁴Catechin hydrate or nother flavonoid, ⁵Emuslifier may be lecithin,sorbitans, polysorbates; ⁶envelope gp120 or influenza hemmagglutininproteins, ⁷neomycin or polymyxin B, ⁸sucrose, lactose or another sugar,⁹Hyaluronic acid or its derivatives (HYAFF) or polyethylene oxidehomopolymers or chitosan.

A composition that includes (50% v/v mustard oil, 0.1% Tween(polysorbate), 50% aqueous phase v/v), the microparticle/micelle size inthe emulsified composition ranges from 0.1 μm-5 μm in size with thelargest at around 5 μm, the smallest at around 0.1 μm, and the majority(70-80%) ranging between 0.5-1 μm.

As used in herein vol/vol refers to the volume of a component in thetotal volume of the adjuvant composition. As used in herein w/w refersto the amount by weight of a component in the total weight of theadjuvant composition.

What is claimed is:
 1. A composition comprising: mustard oil; and atleast one of: (a) a flavonoid, or a flavonoid derivative, or a flavonoidderivative salt; and (b) a vitamin or derivative or salt thereof,wherein said vitamin is selected from the group consisting of vitamin A,vitamin E, vitamin D, vitamin C.
 2. The composition of claim 1, whereinthe composition comprises said flavonoid.
 3. The composition of claim 1,wherein the composition comprises said vitamin.
 4. The composition ofclaim 1, wherein the composition comprises said flavonoid and saidvitamin.
 5. The composition of claim 1, wherein said flavonoid iscatechin.
 6. The composition of claim 1, wherein said vitamin is vitaminE.
 7. The composition of claim 1, wherein said vitamin is vitamin A. 8.The composition of claim 5, further comprising vitamin A.
 9. Thecomposition of claim 1, wherein said vitamin is vitamin C.
 10. Thecomposition of claim 5, wherein said vitamin is vitamin E.
 11. Thecomposition of claim 1, further comprising allyl isothiocyanate.
 12. Thecomposition of claim 1, further comprising an antigen.
 13. A method ofmodulating an immune response in a subject, the method comprisingadministering the adjuvant composition of claim 1 to said subject. 14.The method of claim 13, wherein the subject is a mammal.
 15. The methodof claim 13, wherein said administering is by a route selected from thegroup consisting of: intranasal, pulmonary, sublingual, oral, buccal,intra-vaginal, intra-rectal, ocular, intradermal, and intravascular. 16.A method of making the composition of claim 1, the method comprisingadmixing of said mustard oil and at least one of said flavonoid, or saidflavonoid derivative, or said flavonoid derivative salt; and saidvitamin, or derivative or salt thereof, wherein said vitamin is selectedfrom the group consisting of vitamin A, vitamin E, vitamin D, vitamin C.17. A composition comprising: a pharmaceutically acceptable vegetableoil; a flavonoid, or a flavonoid derivative, or a flavonoid derivativesalt; and a vitamin, or derivative or salt thereof, where the vitamin isselected from the group consisting of vitamin A, vitamin E, vitamin D,vitamin C.
 18. The composition of claim 17, wherein saidpharmaceutically acceptable oil is rubefacient oil.
 19. The compositionof claims 17, wherein said flavonoid is catechin.
 20. The composition ofclaim 17, wherein said vitamin is vitamin E.
 21. The composition ofclaim 17, wherein said vitamin is vitamin A.
 22. The composition ofclaim 17, wherein said vitamin is vitamin C.
 23. The composition ofclaim 19, further comprising vitamin A.
 24. The composition of claim 17,further comprising allyl isothiocyanate.
 25. The composition of claim17, further comprising an antigen.
 26. A method of modulating an immuneresponse in a subject, the method comprising administering the adjuvantcomposition of claim 17 to said subject.
 27. The method of claim 17,wherein the subject is a mammal.
 28. The method of claim 17, whereinsaid administering is by a route selected from the group consisting of:intranasal, pulmonary, sublingual, oral, buccal, ocular, intra-vaginal,intra-rectal, intradermal, and intravascular.
 29. A method of making thecomposition of claim 17, the method comprising admixing of: saidpharmaceutically acceptable oil; said flavonoid, or said flavonoidderivative, or said flavonoid derivative salt; and said vitamin, or aderivative or salt thereof, wherein the vitamin is selected from thegroup consisting of vitamin A, vitamin E, vitamin D, vitamin C.